Treatment of Mycobacterial diseases by administration of bactericidal/permeability-increasing protein products

ABSTRACT

The present invention relates to methods for treating a subject suffering from infection with Mycobacteria, such as  M. leprae  or  M. tuberculosis  comprising administering to the subject a composition comprising a bactericidal/permeability-inducing (BPI) protein product alone or in combination with administration of an anti-Mycobacterial antibiotic.

[0001] This is a continuation-in-part of co-pending U.S. patentapplication Ser. No. 08/031,145, filed Mar. 12, 1993.

BACKGROUND OF THE INVENTION

[0002] The present invention relates to methods of treating a subjectsuffering from infection with Mycobacteria by administration ofBactericidal/Permeability-Increasing Protein (BPI) protein products.Mycobacterium is a non-motile, acid-fast, aerobic, genus of bacteriaknown to cause grave human and animal diseases, such as tuberculosis andleprosy. Infections caused by M. avium are the most common form ofdisseminated bacterial disease in AIDS patients. Orme, et al., Infect.and Immun., 61 (1):338-342 (1993).

[0003] The administration of conventional antibiotics to treatMycobacterial infection is known in the art and has achieved varyingsuccess depending on the susceptibility of the bacterial strain, theefficacy and toxicity of the antibiotic(s) employed, the duration oftreatment, and numerous other factors. Antimicrobials that have beenemployed alone or in combination to treat Mycobacterial infections,including those caused by M. tuberculosis include isoniazid, rifampin,ethambutol, p-aminosalicylic acid, pyrazinamide, streptomycin,capreomycin, cycloserine, ethionamide, kanamycin, amikacin, amithiozone,rifabutin, clofazimine, arithromycin, clarithromycin, ciprofloxacin andofloxacin. McClatchy, Antimycobacterial Drugs: Mechanisms of Action,Drug Resistance, Susceptibility Testing, and Assays of Activity inBiological Fluids, pp. 134-197, In Antibiotics in Laboratory Medicine,3rd ed., V. Lorian, ed. The Williams & Wilkins Co., Baltimore (1991). Asmany Mycobacterial strains are drug resistant, serious obstacles existfor control and successful treatment of tuberculosis and otherMycobacterial diseases. Id.

[0004] A variety of factors have made treatment of individuals afflictedwith Mycobacterial diseases problematic. First, Mycobacteria possess avery hydrophobic cell wall that affords protection against the host'simmune system. As Mycobacterial infections tend to be chronic, thepathologies of these organisms are generally due to host response. Also,many Mycobacterial strains are drug-resistant. These and other factorsmake the development of novel, effective methods for treatingMycobacterial diseases highly desirable.

[0005] Mycobacteria are readily distinguished from gram-negative andgram-positive bacteria by acid fast staining due to significantdifferences in cell wall structure. Gram-negative bacteria arecharacterized by a cell wall composed of a thin layer of peptidoglycancovered by an outer membrane of lipoprotein and lipopolysaccharide(LPS), whereas gram-positive bacteria have a cell wall with a thickerlayer of peptidoglycan with attached teichoic acids, but no LPS. TheMycobacterial cell wall is rich in fatty acids, including a majorconstituent, lipoarabinomannan (LAM), which is widely distributed withinthe cell wall of Mycobacterium species. LAM has been purified from bothM. leprae and M. tuberculosis. Hunter et al., J Biol. Chem.,261:12345-12351 (1986). LAM is a serologically active mannose containingphosphorylated lipopolysaccharide that may be membrane associated.

[0006] The complex physiological effects of LAM appear to beconcentration, time, and source-dependent. For example, Chaterjee etal., Infect. and Immun., 60(3):1249-1253 (1992), reported that, in thefirst 24 hours following exposure, LAM from an avirulent strain oftuberculosis was 100-fold more potent at stimulating TNF secretion inmouse macrophages than LAM from a virulent strain. LAM concentrations of0.01-10 μg/ml for the avirulent strain and 0.01-100 μg/ml for thevirulent strain were tested, and increased LAM concentration wasassociated with increased TNF production with LAM from both species.

[0007] Macrophage-inhibitory effects of LAM have also been described inthe art. LAM purified from both M. leprae and M. tuberculosis has beenreported to be a potent in vitro inhibitor of T-cell lymphokineactivation of mouse macrophages. Sibley et al., Infection and Immunity,56(5):1232-1236 (1988). Because the principle efferent role of themacrophage in acquired resistance to intracellular pathogens requiresactivation by T-cell lymphokines, notably gamma-interferon (IFN-γ),macrophages whose activation-response is inhibited are severelycompromised in their capacity for both enhanced microbicidal andtumoricidal activities.

[0008] In another study, Sibley et al., Clin. Exp. Immunol.,80(1):141-148 (1990), reported that pretreatment of mouse macrophageswith 50 to 100 ug/ml LAM blocked macrophage activation by IFN-γ, butpretreatment with 10 μg/ml LAM did not affect macrophage activation.Thus, it is believed that low concentrations of LAM stimulate cytokineproduction, at least initially. However, higher concentrations of LAM(50-100 μg/ml or more) appear to block rather than promote macrophagefunction. Thus, the production of either too much or too little cytokineat different stages of Mycobacterial disease may contribute toMycobacterial pathogenesis. New methods for blocking theabove-characterized physiological effects of LAM molecules are a highlydesirable goal in the treatment of subjects that are or that have beeninfected with Mycobacteria. For the same reasons, new methods by whichfluids containing LAM can be decontaminated prior to administration intoa subject are also desirable. Neutralization of even small amounts ofLAM is desirable, because small amounts of LAM may have thephysiological effect of stimulating cytokine production.

[0009] Of interest to the background of the invention are thedisclosures of PCT/US88/00510, (WO 88/06038) published Aug. 25, 1988,indicating that certain poloxypropylene/polyoxyethylene nonionicsurface-active block copolymers can be used with or without conventionalantibiotics to treat infection with Mycobacterium. This reference citesstudies suggesting that the effects of other nonionic surfactants ontuberculosis are most likely due to modification of surface lipids ofMycobacteria, and not to direct bactericidal effects on Mycobacteria.See e.g. Cornforth et al., Nature, 168:150-153 (1951).

[0010] Bactericidal/permeability-increasing protein (BPI) is a proteinisolated from the granules of mammalian polymorphonuclear neutrophils(PMN), which are blood cells essential in the defense against invadingmicroorganisms. Human BPI protein has been isolated from PMN's by acidextraction combined with either ion exchange chromatography Elsbach, J.Biol. Chem., 254:11000 (1979) or E. coli affinity chromatography, Weiss,et al., Blood, 69: 652 (1987), and has potent bactericidal activityagainst a broad spectrum of gram-negative bacteria. The molecular weightof human BPI is approximately 55,000 Daltons (55 kD). The amino acidsequence of the entire human BPI protein, as well as the DNA encodingthe protein, have been elucidated in FIG. 1 of Gray, et al., J. Biol.Chem., 264: 9505 (1989), incorporated herein by reference.

[0011] BPI has been shown to be a potent bactericidal agent activeagainst a broad range of gram-negative bacterial species. The cytotoxiceffect of BPI was originally established to be highly specific tosensitive gram-negative species, with no toxicity being noted for othernon-acid fast, gram-positive bacteria or for eukaryotic cells. Theprecise mechanism by which BPI kills bacteria is as yet unknown, but itis known that BPI must first attach to the surface of susceptiblegram-negative bacteria. It is thought that this initial binding of BPIto the bacteria involves electrostatic interactions between the basicBPI protein and negatively charged sites on lipopolysaccharides (LPS).LPS has been referred to as endotoxin because of the potent inflammatoryresponse that it stimulates. LPS induces the release of mediators byhost inflammatory cells which may ultimately result in irreversibleendotoxic shock. BPI binds to Lipid A, the most toxic and mostbiologically active component of LPS.

[0012] In susceptible bacteria, it is thought that BPI binding disruptsLPS structure, leads to an activation of bacterial enzymes that degradephospholipids and peptidoglycans, alters the permeability of the cell'souter membrane, and ultimately causes cell death by an as yet unknownmechanism. BPI is also capable of neutralizing the endotoxic propertiesof LPS to which it binds. Because of its gram-negative bactericidalproperties and its ability to neutralize LPS, BPI can be utilized forthe treatment of mammals suffering from diseases caused by gram-negativebacteria, such as bacteremia or sepsis.

[0013] An approximately 25 kD proteolytic fragment corresponding to theamino-terminal portion of human BPI holoprotein possesses theantibacterial efficacy of the naturally-derived 55 kD human holoprotein.In contrast to the amino-terminal portion the carboxy-terminal region ofthe isolated human BPI protein displays only slightly detectableanti-bacterial activity. Ooi, et al., J. Exp. Med., 174:649 (1991). ABPI amino-terminal fragment, expressed from a construct encodingapproximately the first 199 amino acid residues of the human BPIholoprotein, has been produced by recombinant means as a 23 kD proteinreferred to as “rBPI₂₃”. Gazzano-Santoro et al., Infect. Immun. 60:4754-4761 (1992).

[0014] While BPI protein products are effective for treatment ofconditions associated with gram-negative bacterial infection, therecontinues to exist a need in the art for products and methods fortreatment of other bacterial infections such as infection withMycobacteria.

SUMMARY OF THE INVENTION

[0015] The present invention provides methods of treating a subjectsuffering from infection with Mycobacteria by administration of acomposition comprising a BPI protein product. Therapeutic compositionsaccording to the invention may be administered orally, systemically(such as by intravenous, intramuscular or other injection), or as anaerosol. Mycobacterial disease states subject to treatment according tothe invention include tuberculosis, which can be caused by infectionwith M. tuberculosis, leprosy, which can be caused by infection with M.leprae, and diseases caused by M. avium and other Mycobacteria species.According to preferred methods, anti-Mycobacterial antibiotics such aspreviously identified and/or surfactants may be administered incombination with the BPI protein product to subjects suffering frominfection with Mycobacteria.

[0016] According to another aspect of the present invention,compositions comprising a BPI protein product are administered toneutralize LAM's physiological effects on a host. For example, methodsare provided for neutralizing the effect of low concentrations of LAMcapable of stimulating cytokine production in a host. Methods are alsoprovided for neutralizing the inhibitory effect that higherconcentrations of Mycobacterial LAM (i.e. 100 μg/ml or more) have uponthe interferon-mediated activation of macrophages. Specifically, a BPIprotein product may be administered to an immunosuppressed subjectfailing to respond to microbes or tumor cells due to LAM-inducedinsensitivity of macrophages to activation by T-cell lymphokines.

[0017] According to a further aspect of the present invention, a BPIprotein product is employed in methods for decontaminating a fluidcontaining LAM prior to administration of the fluid into a subject. Suchdecontamination methods of the invention involve contacting the fluidwith the BPI protein product prior to administration, under conditionssuch that LAM forms a complex with the BPI protein product which can beremoved from the fluid. Fluids subject to decontamination by the methodsof this invention include, but are not limited to, blood, plasma, bloodserum, bone marrow, isotonic solutions, pharmaceutical agents, and cellculture agents.

[0018] A further aspect of this invention relates to the use of acomposition comprising a BPI protein product for the manufacture of amedicament for the therapeutic application of treating any of theaforementioned conditions or infections from which a subject mightsuffer.

[0019] Numerous additional aspects and advantages of the invention willbecome apparent to those skilled in the art upon considering thefollowing detailed description of the invention, which describespresently preferred embodiments thereof

BRIEF DESCRIPTION OF THE FIGURES

[0020]FIG. 1 graphically depicts the results of an assay of BPI proteinproduct binding to E. coli J5 Lipid A and M. tuberculosis and FIG. 2graphically represents the results of test to assess the ability of aBPI protein product to inhibit mycobacterial induced TNF production inwhole blood.

DETAILED DESCRIPTION OF THE INVENTION

[0021] The present invention relates to the discovery that a compositioncomprising a BPI protein product can be administered for effectivetreatment of a subject suffering from infection with Mycobacteria. Inparticular, the invention provides methods for treatment of leprosy andtuberculosis, grave diseases caused by the species M. leprae and M.tuberculosis, respectively. It is contemplated that the methodsdescribed herein may be used to treat infection with other Mycobacterialspecies, most notably M. avium and M. intracullulare (collectively knownas “MAC”), but also M. marinum, M. fortuitum, M. chelonae, M. smegmatis,M. kansasii, M. bovis, M. hominis, M. gordonae and other parthogenic oropportunistic species. Beneficial effects of treatment with BPI proteinproducts are expected to result from binding of the products to LAM anddisruption of the bacterial cell wall components (with or without directkilling of the bacteria) in manner similar to that resulting fromtreatment of gram-negative disease states.

[0022] As used herein, “BPI protein product” includes naturally andrecombinantly produced BPI protein; natural, synthetic, and recombinantbiologically active polypeptide fragments of BPI protein; biologicallyactive polypeptide variants of BPI protein or fragments thereof,including hybrid fusion proteins and dimers; and biologically activepolypeptide analogs of BPI protein or fragments or variants thereof,including cysteine-substituted analogs. The BPI protein productsadministered according to this invention may be generated and/orisolated by any means known in the art. U.S. Pat. No. 5,198,541, thedisclosure of which is hereby incorporated by reference, disclosesrecombinant genes encoding and methods for expression of BPI proteinsincluding recombinant BPI holoprotein, referred to herein as rBPI₅₀ andrecombinant fragments of BPI. Co-owned, copending U.S. patentapplication Ser. No. 07/885,501 and a continuation-in-part thereof, U.S.patent application Ser. No. 08/072,063 filed May 19, 1993 which arehereby incorporated by reference, disclose novel methods for thepurification of recombinant BPI protein products expressed in andsecreted from genetically transformed mammalian host cells in cultureand discloses how one may produce large quantities of recombinant BPIproducts suitable for incorporation into stable, homogeneouspharmaceutical preparations.

[0023] Biologically active fragments of BPI (BPI fragments) includebiologically active molecules that have the same amino acid sequence asa natural human BPI holoprotein, except that the fragment molecule lacksamino-terminal amino acids, internal amino acids, and/orcarboxy-terminal amino acids of the holoprotein. Nonlimiting examples ofsuch fragments include an N-terminal fragment of natural human BPI ofapproximately 25 kD, described in Ooi et al., J. Exp. Med., 174:649(1991), and the recombinant expression product of DNA encodingN-terminal amino acids from residue 1 to about residue 200, includingfrom about residue 1 to about residue 193 or 199 of natural human BPI,described in Gazzano-Santoro et al., Infect. Immun. 60:47544761 (1992),and referred to as rBPI₂₃. In that publication, an expression vector wasused as a source of DNA encoding a recombinant expression product(rBPI₂₃) having the 31-residue signal sequence and the first 199 aminoacids of the N-terminus of the mature human BPI, as set out in FIG. 1 ofGray et al., supra, except that valine at position 151 is specified byGTG rather than GTC and residue 185 is glutamic acid (specified by GAG)rather than lysine (specified by AAG). Recombinant holoprotein (rBPI)has also been produced having the sequence (SEQ. ID NOS:1 and 2) set outin FIG. 1 of Gray et al., supra, with the exceptions noted for rBPI₂₃and with the exception that residue 417 is alanine (specified by GCT)rather than valine (specified by GTI). Other examples include dimericBPI forms as described in co-owned and co-pending U.S. patentapplication Ser. No. 08/212,132, filed Mar. 11, 1994, the disclosure ofwhich is hereby incorporated by reference.

[0024] Biologically active variants of BPI (BPI variants) include butare not limited to recombinant hybrid fusion proteins, comprising BPIholoprotein or a biologically active fragment thereof and at least aportion of at least one other polypeptide, and dimeric forms of BPIvariants. Examples of such hybrid fusion proteins and dimeric forms aredescribed by Theofan et al. in co-owned, copending U.S. patentapplication Ser. No. 07/885,911, and a continuation-in-part applicationthereof U.S. patent application Ser. No. 08/064,693 filed May 19, 1993which are incorporated herein by reference in their entirety and includehybrid fusion proteins comprising, at the amino-terminal end, a BPIprotein or a biologically active fragment thereof and, at thecarboxy-terminal end, at least one constant domain of an immunoglobulinheavy chain or allelic variant thereof.

[0025] Biologically active analogs of BPI (BPI analogs) include but arenot limited to BPI protein products wherein one or more amino acidresidue has been replaced by a different amino acid. For example,co-owned, copending U.S. patent application Ser. No. 08/013,801 (Theofanet al., “Stable Bactericidal/Permeability-Increasing Protein Productsand Pharmaceutical Compositions Containing the Same,” filed Feb. 2,1993), the disclosure of which is incorporated herein by reference,discloses polypeptide analogs of BPI and BPI fragments wherein acysteine residue is replaced by a different amino acid. A preferred BPIprotein product described by this application is the expression productof DNA encoding from amino acid 1 to approximately 193 or 199 of theN-terminal amino acids of BPI holoprotein, but wherein the cysteine atresidue number 132 is substituted with alanine and is designatedrBPI₂₁Δcys or rBPI₂₁.

[0026] Other BPI protein products useful according to the methods of theinvention are peptides derived from or based on BPI produced byrecombinant or synthetic means (BPI-derived peptides), such as thosedescribed in co-owned and copending U.S. patent application Ser. No.08/209,762, filed Mar. 11, 1994, which is a continuation-in-part of U.S.patent application Ser. No. 08/183,222, filed Jan. 14, 1994, which is acontinuation-in-part of U.S. patent application Ser. No. 08/093,202filed Jul. 15, 1993), which is a continuation-in-part of U.S. patentapplication Ser. No. 08/030,644 filed Mar. 12, 1993, the disclosures ofwhich are hereby incorporated by reference. Other useful BPI proteinproducts include peptides based on or derived from BPI which aredescribed in co-owned and co-pending U.S. patent application Ser. No.08/274,299 filed Jul. 11, 1994, by Horwitz et al. and U.S. patentapplication Ser. No. 08/273,540, filed Jul. 11, 1994, by Little et al.

[0027] Presently preferred BPI protein products includerecombinantly-produced N-terminal fragments of BPI, especially thosehaving a molecular weight of approximately between 21 to 25 kD such asrBPI₂₁ or rBPI₂₃, dimeric forms of these N-terminal fragments.Additionally, preferred BPI protein products include rBPI₅₀ andBPI-derived peptides.

[0028] The administration of BPI protein products is preferablyaccomplished with a pharmaceutical composition comprising a BPI proteinproduct and a pharmaceutically acceptable diluent, adjuvant, or carrier.The BPI protein product may be administered without or in conjunctionwith known surfactants, other chemotherapeutic agents. A preferredpharmaceutical composition containing BPI protein products comprises theBPI protein product at a concentration of 1 mg/ml in citrate bufferedsaline (5 or 20 mM citrate, 150 mM NaCl, pH 5.0) comprising 0.1% byweight of poloxamer 188 (Pluronic F-68, BASF Wyandotte, Parsippany,N.J.) and 0.002% by weight of polysorbate 80 (Tween 80, ICI AmericasInc., Wilmington, Del.). Another preferred pharmaceutical compositioncontaining BPI protein products comprises the BPI protein product at aconcentration of 2 mg/ml in 5 mM citrate, 150 mM NaCl, 0.2% poloxamer188 and 0.002% polysorbate 80. Such preferred combinations are describedin co-owned, co-pending, U.S. patent application Ser. No. 08/190,869filed Feb. 2, 1994 (McGregor et al., “Improved PharmaceuticalCompositions”), and U.S. patent application Ser. No. 08/012,360 filedFeb. 2, 1993 (McGregor et al., “Improved Pharmaceutical Composition”),the disclosures of which are incorporated herein by reference.

[0029] The BPI protein product can be administered by any known method,such as orally, systemically (such as by intravenous, intramuscular orother injection), or as an aerosol. Medicaments can be prepared for oraladministration or by injection or other parenteral methods andpreferably include conventional pharmaceutically acceptable carriers andadjuvents as would be known to those of skill in the art. Themedicaments may be in the form of a unit dose in solid, semi-solid andliquid dosage forms such as tablets, pills, powders, liquid solutions orsuspensions, and injectable and infusible solutions. Effective dosageranges from about 100 μg/kg to about 10 mg/kg of body weight arecontemplated. Intravenous administration is a preferred method fortreatment of leprosy.

[0030] It is contemplated that aerosol administration to the lungs willbe a preferred method for treating other Mycobacterial infections, suchas tuberculosis. Such aerosol formulations would be manufactured bymeans that are known in the art, and administered by metered-doseinhaler, updraft nebulization, or other means known in the art.

[0031] An aspect of the present invention is to provide methods oftreating a subject suffering from any of the physiological effects ofMycobacterial LAM. As described above, the physiological effects of LAMdepend on a number of factors, including the source and concentration ofthe LAM, and the length of time to which host cells are exposed to LAM.Example 3, infra, demonstrates that 20-100 μg/ml of nonviable,desiccated M. tuberculosis added to whole blood will stimulate TNFproduction by the monocytes in the blood. Other studies described abovehave shown that 50-100 μg/ml of LAM will down-regulate macrophagefunctions and expression (TNF, and the like) and prevent macrophageactivation, said methods comprise administering a BPI protein product tothe subject. Methods are provided for treating a subject suffering fromthe effects of increased cytokine production caused by the physiologicalpresence of LAM. Methods are also provided for treating a subjectsuffering from LAM-induced inhibition of macrophage activation, and theeffects thereof. Methods and formulations by which a BPI protein productmay be administered, including preferred methods and formulations, arethe same as those set forth above for the treatment of Mycobacterialinfection.

[0032] Because of the harmful physiological effects that MycobacterialLAM can have on a subject, even in the absence of viable Mycobacteria,methods are provided in the present invention by which a fluidcontaining LAM may be decontaminated prior to administration of thefluid into a subject. Such methods comprise contacting the fluid with aBPI protein product prior to administration, under conditions such thatLAM forms a complex with the BPI protein product, therebydecontaminating the fluid. By way of nonlimiting examples, such methodsmay be applied to fluids such as blood, plasma, blood serum, bonemarrow, isotonic solutions, pharmaceutical agents, or cell culturereagents.

[0033] BPI protein product is thought to interact with a variety of hostdefense elements present in whole blood or serum, including complementand LBP, and other cells and components of the immune system. Suchinteractions might result in potentiating and/or synergizing theanti-microbial activities. Because of these interactions, BPI proteinproducts are expected to exert even greater activity in vivo than invitro. Thus, while in vitro tests are predictive of in vivo utility,absence of activity in vitro does not necessarily indicate absence ofactivity in vivo. For example, BPI has been observed to display agreater bactericidal effect on certain gram-negative bacteria in wholeblood or plasma assays than in assays using conventional media. [Weisset al., J. Clin. Invest. 90:1122-1130 (1992)]. This may be becauseconventional in vitro systems lack the blood elements that facilitate orpotentiate BPI's function in vivo, or because conventional mediadesigned to maximize bacterial growth contain higher than physiologicalconcentrations of magnesium and calcium, inhibitors of BPI proteinproduct antibacterial activity.

[0034] Therapeutic effectiveness is based on a successful clinicaloutcome, and does not require that an anti-mycobacterial agent or agentskill 100% of the organism involved in the infection. Frequently,reducing organism load by one log (factor of 10) permits the host's owndefenses to control the infection. In addition, augmenting an earlyanti-mycobacterial effect can be particularly important in addition toany long-term anti-mycobacterial effect. These early events are asignificant and critical part of therapeutic success, because they allowtime for host defense mechanisms to activate.

[0035] It is also contemplated that the BPI protein product beadministered with other products that potentiate the anti-mycobacterialactivity of BPI protein products. For example, serum complementpotentiates the gram-negative bactericidal activity of BPI proteinproducts; the combination of BPI protein product and serum complementprovides synergistic bactericidal/growth inhibitory effects. See, e.g.,Ooi et al. J. Biol. Chem., 265: 15956 (1990) and Levy et al. J. Biol.Chem., 268: 6038-6083 (1993) which address naturally-occurring 15 kDproteins potentiating BPI antibacterial activity. See also co-owned,co-pending U.S. patent application Ser. No. 08/093,201 filed Jul. 14,1993, and continuation-in-part, U.S. patent application Ser. No.08/274,303 filed Jul. 11, 1994 which describes methods for potentiatinggram-negative bactericidal activity of BPI protein products byadministering lipopolysaccharide binding protein (LBP) and LBP proteinproducts. The disclosures of these applications are incorporated byreference herein. LBP protein derivatives and derivative hybrids whichlack CD-14 immunostimulatory properties are described in co-owned,co-pending U.S. patent application Ser. No. 08/261,660, filed Jun. 17,1994 as a continuation-in-part of U.S. patent application Ser. No.08/079,510, filed Jun. 17, 1993, the disclosures of which areincorporated by reference herein.

[0036] An aspect of this invention includes the use of a compositioncomprising a BPI protein product for the manufacture of a medicament forthe therapeutic application of treating any of the aforementionedconditions or diseases from which a subject suffers. The medicament mayinclude, in addition to a BPI protein product, other chemotherapeuticagents such as known anti-mycobacterial antibiotics or surfactants. Themedicament may additionally or alternatively include one or moreadditional pharmaceutically acceptable components, such as diluents,adjuvants, or carriers.

[0037] An aspect of the present invention is the ability to provide moreeffective treatment of Mycobacterial infection by virtue of thesynergistic increase in or potentiation of the anti-bacterial activitiesof an anti-Mycobacterial antibiotic or BPI protein product. Aspreviously noted, anti-Mycobacterial antibiotic therapy currentlyinvolves administration of one or more (and frequently three or more)antibiotics such as isoniazid, rifampin, ethambutol, p-aminosalicylicacid, pyrazinamide, streptomycin, capreomycin, cycloserine, ethionamide,kanamycin, amikacin, amithiozone, rifabutin, clofazimine, arithromycin,clarithromycin, ciprofloxacin and ofloxacin. Unlike some therapeuticagents, BPI protein product is easily administered and produces noinflammatory reaction. An aspect of the present invention is the abilityto treat Mycobacterial organisms that are normally resistant to one ormore antibiotics. A further aspect is the ability to use lowerconcentrations of relatively toxic or expensive antibiotics such asrifampin. Because the use of some antibiotics is limited by theirsystemic toxicity or prohibitive cost, lowering the concentration ofantibiotic required for therapeutic effectiveness reduces toxicityand/or cost of treatment, and thus allows wider use of the antibiotic.The present invention may also provide quality of life benefits due to,e.g., decreased duration of therapy, reduced stay in intensive careunits or overall in the hospital, with the concomitant reduced risk ofserious nosocomial (hospital-acquired) infections.

[0038] The invention further provides pharmaceutical compositions fortreatment of Mycobacterial infection and the sequelae thereof comprisingthe combination of a BPI protein product and an antibiotic which ispresent in an amount effective to have synergistic or potentiatingbactericidal/bacteriostatic properties, including increasedsusceptibility or reversal of resistance. The pharmaceutical compositioncan comprise a pharmaceutically-acceptable diluent, adjuvant or carrier.

[0039] Methods of the present invention are more fully illustrated bythe nonlimiting examples which follow. Example 1 address BPI proteinproducts binding to a species of Mycobacterium, M. tuberculosis. Example2 address prospective use of BPI protein products in binding purifiedLAM of Mycobacteria. Examples 3 and 4 describe attempts to reverseMycobacteria-induced cytokine production in whole human blood. Example 5addresses use of BPI protein products in combination withanti-mycobacterial antibiotics to inhibit M. tuberculosis growth.Remaining Examples 6-13 address prospective in vitro and in vivo use ofBPI protein products according to methods of this invention. The modelsdescribed in those examples and/or other models known in the art areused to predict the efficacy and the optimal BPI protein productformulations of the methods of invention.

EXAMPLE 1

[0040] An enzyme linked immunosorbent assay (ELISA) was conducted todetermine binding of a BPI protein product to M. tuberculosis.Specifically, non-viable, desiccated M. tuberculosis H37 RA (Difco,Detroit Mich.) was suspended in DPBS (25 μg/ml) and used to coatmicrotiter wells overnight at 37° C. Wells were also coated with either25 μg/ml Lipid A (E. coli J5 mutant, RIBI, Hamilton Mont.) or 500 μlDPBS to demonstrate the functionality and specificity of rBPI₂₃. Afterwashing (3× with DPBS+0.05% Tween 20), the plates were blocked for 1 hr.at room temperature with 200 μl/well of DPBS+1% non-fat milk. Afterwashing as above, 50 μl solutions of either various concentrations ofrBPI₂₃ (in DPBS containing 0.05% Tween 20) or DPBS (negative control)were added to the wells, which were then incubated for 1 hr. at 37° C.The wells were again washed as above, and the amount of rBPI₂₃ bound tothe wells was determined using an anti-rBPI₂₃ mouse monoclonal antibody(designated αBPI MAb-2-4) and an enzyme conjugated anti-murine IgGantibody (HRP-Ab, Zymed #61-0120, San Francisco, Calif.). To each well100 μl of aBPI MAb-2-4 was added (100 ng/ml in DPBS+0.05% Tween 20), andthe plates were incubated 1 hr. at 37° C. After washing as above, 100 μlof HRP-Ab was added (1:1000 in DPBS+0.05% Tween 20) to each well and theplates were again incubated 1 hr. at 37° C. After washing the plates asabove, 100 μl substrate in 0. 1M citrate plus 1:50 ABTS (20 mg/ml stock)and 1:1000 H₂O₂ was added to each well. The plates were incubated 10-30min. at room temperature, and absorbance readings were taken at 405 nm(OD 405).

[0041] The results of the experiment are represented graphically in FIG.1, which depicts the ability of varying concentrations rBPI₂₃ to bind toJ5 Lipid A (filled triangles); to M. tuberculosis (open squares); and tothe no antigen-free control (filled circles). The abscissa of eachmeasurement represents the concentration of rBPI₂₃, and the ordinaterepresents the average OD 405 measurements from four trials. Error barsreflect the variation in OD 405 readings for each data point.

[0042] This experiment demonstrated that rBPI₂₃ binds specifically tonon-viable desiccated M. tuberculosis. The functionality of the rBPI₂₃used in these experiments was confirmed by the results of the Lipid A(positive control) binding assay, and the specificity of the experimentswas confirmed by the lack of binding to the negative control samples.

EXAMPLE 2

[0043] In this example, an ELISA Assay is conducted to determine bindingof a BPI protein product to the lipoarabinomannan portion ofMycobacteria. The binding activity of BPI protein product (e.g., rBPI₂₃)to LAM is demonstrated as described in the previous example, except LAMpurified from a species of Mycobacterium, (e.g., M. tuberculosis or M.leprae) is substituted for the nonviable M. tuberculosis used to coatthe ELISA plates in that example. Purified LAM is isolated as describedby Hunter et al., J. Biol. Chem., 261:12345-12351 (1986). Specificbinding of biologically active BPI protein product is demonstrated bycomparison of the OD 405 readings from the LAM coated wells withpositive and negative controls.

EXAMPLE 3

[0044] The following experiment was conducted to determine the effect ofa BPI protein product, rBPI₂₃, on Mycobacteria-induced cytokineproduction in whole human blood. Whole human blood from healthyvolunteers was collected into Vacutainer tubes (ACD, Beckton Dickinson,Rutherford, N.J.), Aliquots of blood (225 μl) were mixed with eitherrBPI₂₃ (10 μg/ml final) or the protein thaumatin (10 μg/ml final in 5ml) as a negative control. RPMI medium (20 μl) was added to each sample.Varying dilutions (0-8 ng/ml) of either E. coli O113 LPS (Ribi, HamiltonMont.) or of non-viable, desiccated M. tuberculosis H37 RA (0-100 μg/ml)(Difco, Detroit Mich.) were added to the samples, which were thenincubated at 37° C. for 6 hours. The reactions were stopped by theaddition of 750 μl of RPMI medium, the samples were centrifuged at 500 gfor 7 min, and stored at −20° C. until analyzed. The supernatant wasassayed for cytokine (TNF) levels based on a standard curve, accordingto the manufacturers' recommendation (Biokine ELISA test, T CellSciences, Cambridge, Mass.).

[0045] The assay results revealed that rBPI₂₃ at 10 μg/ml had noinhibitory effect on M. tuberculosis-induced TNF release at theconcentration (20-100 μg/ml) of M. tuberculosis added to the bloodsamples. The same concentration of rBPI₂₃ eliminated LPS-induced TNFrelease at the LPS concentrations tested (2-8 ng/ml). The lack ofinhibitory effect on cytokine induction by M. tuberculosis may be theresult of use of sub-optimal dosage levels. Alternatively, somecomponent of the Mycobacterial cell wall other than the LAM bound byrBPI₂₃ may be responsible for inducing cytokine production at theMycobacterium concentrations tested.

EXAMPLE 4

[0046] In this example, multiple additional assays were conducted toassess the inhibitory effect of BPI protein products on mycobacterial(M. tuberculosis or M. smegmatis) induced production of tumor necrosisfactor (TNF) by monocytes/macrophages present in whole human blood.Briefly summarized, live or heat killed mycobacteria at varyingconcentrations was added to whole blood and incubated with either afixed amount BPI protein product or acetate buffer negative controlsolution. After incubation, the content of TNF present was assessed bystandard means. The TNF content of BPI protein product treated sampleswas then compared to the TNF content of buffer control samples todetermine the relative inhibitory effect of the BPI protein producttested. Whole blood samples were obtained from healthy human volunteersand aliquoted as in Example 3. To each tube containing 225 μl of wholeblood was added from 0 to 1×10⁷ live or heat killed mycobacteria.Depending on whether one or two BPI protein products were to be tested,four or six tubes were prepared at each concentration of bacteria, afterwhich the tubes were incubated at 37° C. for 15 minutes. To two of thetubes at each bacterial concentration was added either a selected BPIprotein product at a final concentration of 4 μg/ml or acetate buffer(as a negative control), after which is tubes were further incubated at37° C. for 5 to 6 hours. Thereafter 750 μl of RPMI 1640 was added toeach sample and the samples were centrifuged at 17,000 rpm (500 g?) for6 minutes. Supernatants were stored at −70° C. until thawed immediatelyprior to testing for TNF content using the Biokine ELISA test kit as inExample 3.

[0047] In a first series of assays, heat-killed M. tuberculosis (strainH37Ra) was employed as the TNF stimulating organism. In a first test onwhole blood, no substantial increased in TNF levels was observed untilbacteria were added at a concentration of 1×10⁶ organisms and thepresence of rBPI₂₃ in the samples resulted in an approximately 70%inhibition of TNF production. In a second test involving two separatewhole blood assays, inhibitory effects of both rBPI₂₃ and BPIholoprotein were assessed. Substantial increases in TNF concentrationover basal levels (no microorganisms added) were observed commencing atmicroorganism concentrations of 3×10⁵ up through 1×10⁷. Overall,inhibition of TNF production by 20% or greater was observed when rBPI₂₃was added at all such organism levels. Lesser degrees of inhibition werenoted for the BPI holoprotein (with no inhibition at all noted in oneduplicate test at the highest concentration of organisms). The lessereffects of the holoprotein in these assays are likely attributable tothe lower molar concentration employed. A third M. tuberculosis test wasperformed on whole blood drawn from four different volunteers, usingrBPI₂₃ as the test compound. Expectedly, the level of inhibition of TNFformation by the uniform dose of BPI protein product varied from subjectto subject. With the exception of one subject's blood samples (whereininhibition was observed only at intermediate microorganism concentrationof 1×10⁶ and not at all at concentrations of 1×10⁷), the BPI proteinproduct provided for at least about 10% and up to about 50% TNFinhibition, with higher inhibitory levels being observed at highermicroorganism concentrations. A fourth test involving M. tuberculosiswas carried out using rBPI₂₃ at 4 μg/ml and 8 μg/ml concentrations. TheBPI protein product was again observed to inhibit TNF at the 4 μg/mllevel, with the greatest effects being observed at microorganismconcentrations of 3×10⁶. Doubling the concentration of test compound to8 μg/ml did not enhance, and in fact somewhat diminished, inhibitoryeffects observed.

[0048] In a second series of assays, heat killed M. smegmatis wasemployed to stimulate TNF production in whole blood. In a first test,whole blood from eight different patients was employed and was subjectedto contact with concentrations of 0, 0.5×10⁵, 1×10⁵, 1×10⁶ and 1×10⁷organisms. FIG. 2 provides a graphic representation of the sum of theresults observed and indicated that the rBPI₂₃ product tested at 4 μg/mlwas an effective inhibitor of TNF production at all bacterialconcentrations. In a second test involving blood from two differentsubjects, TNF production inhibitory effects of rBPI₂₃ were assessed forM. smegmatis, E. coli and S. aureus. Expectedly, significant TNFinhibitory effects were observed in the E. coli treated blood, with thegreatest present with microorganism concentrations of 1×10⁵ and thelesser effects at higher concentrations of organisms. Similarly, nosubstantial TNF inhibitory effects were observed for the BPI proteinproduct in the S. aureus assay. Variable results were seen in the M.smegmatis assay; pronounced inhibitory effects were observed in onesubject's blood at the 1×10⁷ concentration of organisms, whileinhibition was observed in the other blood sample only at the 1×10⁶microorganism concentration.

[0049] The above assay results demonstrate in vitro effectiveness of BPIprotein products in inhibiting induction of tumor necrosis factor bymycobacterial species and are predictive of in vivo efficacy in humanpatients.

EXAMPLE 5

[0050] In this example, rBPI₂₃ at varying concentrations was assessedfor its growth inhibitory effect on M. tuberculosis treated with varyingconcentrations of the anti-Mycobacterial antibiotics isoniazid (INH) andrifampin (RMP). Briefly summarized, pure cultures of M. tuberculosis(MTB) were incubated for 24 hours with varying concentrations of rBPI₂₃and antibiotic. Cultures were added to Bactec® bottles (JohnstonLaboratories, Cockeysville, Md.) containing ¹⁴C labeled nutrients anddaily “growth index” values were determined accordingly to thesupplier's instructions on the basis of ¹⁴CO₂ evolved from the medium.In separate assays, concentrations of rBPI₂₃ of 0, 3.9, 15.6, 62.5, 250and 1000 μg/ml were combined with INH at levels of 0, 0.006, 0.012,0.025 and 0.05 μg/ml or RMP levels of 0, 0.12, 0.25, 0.5 and 1.0 μg/ml.Growth index values were assessed daily starting the second day afterinoculation into the vials through to the eighteenth day. In the INHassay, no BPI protein product was added, growth index valuescharacteristically gradually increased over time and as a function ofthe dosage of antibiotic employed (increases generally began earlier androse more steeply at lower doses than at higher doses). Addition ofrBPI₂₃ had variable effects in enhancing or diminishing antibioticeffects on growth index values, depending on the concentration employed.An intermediate dose (62.5 μg/ml) of BPI protein product consistentlytended to reduce growth index values at all doses of INH tested and thusoperated to enhance INH growth inhibitory effects. Similar but lesspronounced enhancement effects were observed for the 15.6 μg/ml rBPI₂₃dose. Lower (0 and 3.9 μg/ml) and higher (250 and 1000 μg/ml) doses ofthe BPI protein product generally diminished the antibiotic effects ofINH, with the highest rBPI₂₃ dose invariably functioning to increasing“swamp out” INH effects on growth index toward the middle of the testperiod. At later times in the test period, however, the highest doses ofBPI protein product appeared to suppress and actually reverse theabove-noted characteristic increases in growth index over time. Incombinative assay with RMP, rBPI₂₃ had no discernible enhancing effectat the highest doses of the antibiotic 0.5 and 1.0 μg/ml where there wasessentially no increase in growth index throughout the entire testperiod. At lesser concentrations of RMP, there tended to be adose-dependent enhancement effect of the BPI protein product, with thegreatest degree of enhancement occurring at the highest doses of rBPI₂₃and no evidence of the “swamp out” effects observed for combinationswith INH intermediate times within the test period.

[0051] The results set out above establish utility of BPI proteinproduct concurrently filed in enhancing the growth inhibitory effects ofanti-mycobacterial antitiotics.

EXAMPLE 6

[0052] The following experiment is conducted to determine the in vitroinhibitory effect of a BPI protein product on the growth of aMycobacterium species, Mycobacterium tuberculosis (MTB). The procedurecan be performed with other cultivable Mycobacterial species and employsconcentrations of a BPI protein product that would be readily generatedin human serum by ordinary modes of oral or parenteral administrationand/or readily delivered to lung surface by aerosol administration. Theeffects of the BPI protein product can be evaluated with and withoutnon-ionic surfactants, and/or standard antibiotics.

[0053] Log phase cultures of antibiotic-sensitive andantibiotic-resistant MTB are incubated in either 7H11 broth medium orwhole human blood, to which the following is added: (a) nothing; (b)surfactant; (c) standard MTB antibiotic; (d) antibiotic plus surfactant.Cultures are incubated with varying concentrations of, e.g., rBPI₂₃.Duplicate cultures grown in each medium are also left untreated byrBPI₂₃ as a negative control. The organisms are placed in Bactec®bottles (Johnston Laboratories, Cockeysville, Md.) containing ¹⁴Clabeled nutrients. rBPI₂₃ challenged M. tuberculosis growth isdetermined by measuring the elution of ¹⁴CO₂ from the medium, comparedto the appropriate negative control. The absence of the formation ¹⁴CO₂by the treated cultures is indicative of the inhibitory affects ofrBPI₂₃ to MTB. Differential amounts of ¹⁴CO₂ formed in the absence orpresence of standard MTB antibiotics and/or surfactants is indicative ofthe synergistic or additive effect that a BPI protein product has whenused conjunctively with such agents. By comparing the results of thisexperiment performed with varying concentrations of the BPI proteinproduct, the effective concentration of the BPI protein product isoptimized. Radiometric assays to test the susceptibility ofMycobacterial species to drugs have been described previously. SeeMcClatchy (cited supra) and references therein.

EXAMPLE 7

[0054] The following experiment is conducted to determine the in vitroeffects of a BPI protein product (rBPI₂₃) in an M. leprae model. Apalmitic acid oxidation assay is used to measure the “viability” of theuncultivable leprosy bacillus adhered to filter paper and “grown” in a¹⁴C-palmitic acid-containing medium. In this method ¹⁴CO₂ evolved fromthe metabolism by M. leprae of ¹⁴C-palmitic acid is trapped on filterpaper moistened with NaOH and radioactivity is determined with a liquidscintillation counter. Susceptibility to BPI protein productformulations is determined by differences in radioactivity for M. lepraetested with such formulations and treated control cultures.

EXAMPLE 8

[0055] The following experiment, which is a variation of an assayconducted by Mittal et al., J. Clin. Microbiol., 17(4):704-707 (1983),is conducted to determine the in vitro inhibitory effect of BPI proteinproduct on the growth of Mycobacterium leprae. The effects of differentconcentrations of BPI protein product on M. leprae are evaluated withand without non-ionic surfactants, and/or standard antibiotics. Theprocedures as described by Mittal et al. are outlined below.

[0056] Skin biopsy specimens from lepromatous patients are homogenizedand are used to inoculate suspensions of mouse peritoneal macrophagescultured in RPMI 1640 (GIBCO Biocult, Irvine, Scotland) enriched with30% fetal calf serum. After incubating 18 hours, fresh media containing[methyl-³H]-thymidine (Amersham International Ltd., Arlington Heights,Ill.) is added and the cultures are incubated for 14 days. The procedureof Mittal et al. is varied by testing the effect of differentconcentrations of BPI protein product with or without surfactants and/orantibiotics on ³H-thymidine incorporation. Macrophages containingphagocytosed viable M. leprae will incorporate ³H-thymidine at a 2 to10-fold higher rate than control cultures containing heat killed M.leprae. Greater than 50% inhibition of ³H-thymidine-incorporation isindicative of bactericidal efficacy of the test product.

EXAMPLE 9

[0057] An experiment is conducted to determine the in vivo effect that aBPI protein product will have on M. tuberculosis species. The modelemployed is a variation of that used by Lalande et al., AntimicrobialAgents and Chemotherapy, 37(3):407-413 (1993), to assess the efficacy ofantimicrobial agents against M. tuberculosis. Mice inoculatedintravenously with M. tuberculosis are treated with various BPI proteinproduct doses alone or in combination with surfactants and/orantibiotics. The efficacy of such treatment regiments is analyzed asdescribed.

EXAMPLE 10

[0058] The following experiment is conducted to determine the effectthat a BPI protein product will have on M. leprae in vivo. The model tobe used is a variation of that developed by Shepard to study the effectof compounds on the growth of M. leprae in the footpads of infectedmice. Shepard et al., Proc. Soc. Exp. Biol. Med., 109:636-638 (1962);Shepard, J. Exp. Med. 112:445-454 (1960). Briefly, leprosy bacilli areinoculated into foot-pads of mice, which are subsequently treated withdifferent amounts of test compound with or without known antibioticsand/or surfactants. Untreated infected mice are used as a control. Micefrom each treatment regimen are sacrificed at monthly intervals, andsections cut from the infected foot. The presence of an area containingacid-fast bacteria can be observed microscopically and/or the number ofsuch bacteria can be counted. See Shepard and McRae, Int. J. Lepr.,36:78-82. Differences between M. leprae bacteria levels observed intreated versus control mice is indicative of the bacteriostatic orbactericidal efficacy of a given BPI treatment regimen. The metabolicstatus of isolated M. leprae may also be measured. Franzblau andHastings, Antimicrobial Agnes and Chemotherapy, 31(5):780-783 (1987).

EXAMPLE 11

[0059] The following experiments are designed to demonstrate that BPIprotein product is able to inhibit the ability of low concentration ofLAM to induce cytokines, yet reverse the unresponsive state that attendshigher concentrations of LAM. Increasing concentrations of LAM arepretreated with BPI protein product at varying concentrations. Thesecomplexes are applied to peritoneal macrophages from normal andMycobacterium species infected mice. TNF production by treated cellswill be assessed.

EXAMPLE 12

[0060] A variation of the armadillo model developed by Kirchheimer etal., Int. J. Lepr., 39:693-702 (1971); Id., 40:229 (1972), is employedto study the in vivo effect of BPI protein product test compositions onthe growth of M. leprae in infected armadillos. Briefly, leprosy bacilliare inoculated into armadillos, which are subsequently treated withdifferent amounts of a test composition. The test compositions willcomprise a BPI protein product, e.g. rBPI₂₃, with or without knownantibiotics and/or surfactants. Untreated infected specimens are used asa control. Armadillos from each treatment regimen are examined andbiopsy specimens analyzed by procedures known in the art. M. lepraeisolated from armadillos is assayed for metabolic activity. Differencesbetween the appearance of lesions, differences in M. leprae bacterialconcentrations, and differences in the metabolic activity of M. lepraeisolates in treated versus control specimens are indicative of thebacteriostatic or bactericidal efficacy of a given BPI treatmentregimen.

EXAMPLE 13

[0061] The following experiment is conducted to determine the level ofdecontamination of a fluid containing LAM that can be achieved bytreatment with a BPI protein product. Whole human blood, plasma, bloodserum or the like is passed through a column containing a matrix, towhich a BPI protein product is bound. Such matrix may be constructed byany means known to those skilled in the art. LAM in the fluid complexeswith the BPI protein product affixed to the matrix as the fluid ispassed through the column. The absence of LAM in the fluid eluted fromthe column demonstrates the effectiveness of a BPI protein product atdecontaminating a fluid containing LAM.

[0062] Alternatively, monoclonal antibodies with binding specificity fora BPI protein product, such as the antibodies employed in Example 1, areaffixed to the matrix. A sufficient amount of a BPI protein product isadded to the mixture to bind any LAM present in the fluid. The fluid ispurified by passing it through the column. The αBPI antibodies affixedto the column bind the LAM/BPI protein product complex in the fluid, andthe fluid eluted from the column is analyzed for the presence or absenceof LAM contamination.

[0063] Numerous modifications and variations in the practice of theinvention are expected to occur to those skilled in the art uponconsideration of the foregoing description of the presently preferredembodiments thereof. For example, while the above illustrative examplesprincipally address studies predictive of antibacterial effects in thecontext M. tuberculosis and M. leprae, model studies of infection with,e.g., M. avium [see, e.g., Brown et al., Antimicrob. Agents and Therapy,37(3): 398-402(1993)] are also expected to reveal effectiveness of BPIprotein product therapies. As another example, preliminary experimentaldata indicates that BPI protein products alone and/or in combinationwith cytokines such as gamma interferon (and in combination withantibiotics as well) can enhance the rate at which human monocytesphagocytize Mycobacterial organisms. Combinative therapies involvingadministration of cytokines along with BPI protein products (andantibiotics) are thus within the scope of the invention). Consequently,the only limitations which should be placed upon the scope of thepresent invention are those which appear in the appended claims.

1 2 1813 base pairs nucleic acid single linear cDNA CDS 31..1491mat_peptide 124..1491 1 CAGGCCTTGA GGTTTTGGCA GCTCTGGAGG ATG AGA GAG AACATG GCC AGG GGC 54 Met Arg Glu Asn Met Ala Arg Gly -31 -30 -25 CCT TGCAAC GCG CCG AGA TGG GTG TCC CTG ATG GTG CTC GTC GCC ATA 102 Pro Cys AsnAla Pro Arg Trp Val Ser Leu Met Val Leu Val Ala Ile -20 -15 -10 GGC ACCGCC GTG ACA GCG GCC GTC AAC CCT GGC GTC GTG GTC AGG ATC 150 Gly Thr AlaVal Thr Ala Ala Val Asn Pro Gly Val Val Val Arg Ile -5 1 5 TCC CAG AAGGGC CTG GAC TAC GCC AGC CAG CAG GGG ACG GCC GCT CTG 198 Ser Gln Lys GlyLeu Asp Tyr Ala Ser Gln Gln Gly Thr Ala Ala Leu 10 15 20 25 CAG AAG GAGCTG AAG AGG ATC AAG ATT CCT GAC TAC TCA GAC AGC TTT 246 Gln Lys Glu LeuLys Arg Ile Lys Ile Pro Asp Tyr Ser Asp Ser Phe 30 35 40 AAG ATC AAG CATCTT GGG AAG GGG CAT TAT AGC TTC TAC AGC ATG GAC 294 Lys Ile Lys His LeuGly Lys Gly His Tyr Ser Phe Tyr Ser Met Asp 45 50 55 ATC CGT GAA TTC CAGCTT CCC AGT TCC CAG ATA AGC ATG GTG CCC AAT 342 Ile Arg Glu Phe Gln LeuPro Ser Ser Gln Ile Ser Met Val Pro Asn 60 65 70 GTG GGC CTT AAG TTC TCCATC AGC AAC GCC AAT ATC AAG ATC AGC GGG 390 Val Gly Leu Lys Phe Ser IleSer Asn Ala Asn Ile Lys Ile Ser Gly 75 80 85 AAA TGG AAG GCA CAA AAG AGATTC TTA AAA ATG AGC GGC AAT TTT GAC 438 Lys Trp Lys Ala Gln Lys Arg PheLeu Lys Met Ser Gly Asn Phe Asp 90 95 100 105 CTG AGC ATA GAA GGC ATGTCC ATT TCG GCT GAT CTG AAG CTG GGC AGT 486 Leu Ser Ile Glu Gly Met SerIle Ser Ala Asp Leu Lys Leu Gly Ser 110 115 120 AAC CCC ACG TCA GGC AAGCCC ACC ATC ACC TGC TCC AGC TGC AGC AGC 534 Asn Pro Thr Ser Gly Lys ProThr Ile Thr Cys Ser Ser Cys Ser Ser 125 130 135 CAC ATC AAC AGT GTC CACGTG CAC ATC TCA AAG AGC AAA GTC GGG TGG 582 His Ile Asn Ser Val His ValHis Ile Ser Lys Ser Lys Val Gly Trp 140 145 150 CTG ATC CAA CTC TTC CACAAA AAA ATT GAG TCT GCG CTT CGA AAC AAG 630 Leu Ile Gln Leu Phe His LysLys Ile Glu Ser Ala Leu Arg Asn Lys 155 160 165 ATG AAC AGC CAG GTC TGCGAG AAA GTG ACC AAT TCT GTA TCC TCC AAG 678 Met Asn Ser Gln Val Cys GluLys Val Thr Asn Ser Val Ser Ser Lys 170 175 180 185 CTG CAA CCT TAT TTCCAG ACT CTG CCA GTA ATG ACC AAA ATA GAT TCT 726 Leu Gln Pro Tyr Phe GlnThr Leu Pro Val Met Thr Lys Ile Asp Ser 190 195 200 GTG GCT GGA ATC AACTAT GGT CTG GTG GCA CCT CCA GCA ACC ACG GCT 774 Val Ala Gly Ile Asn TyrGly Leu Val Ala Pro Pro Ala Thr Thr Ala 205 210 215 GAG ACC CTG GAT GTACAG ATG AAG GGG GAG TTT TAC AGT GAG AAC CAC 822 Glu Thr Leu Asp Val GlnMet Lys Gly Glu Phe Tyr Ser Glu Asn His 220 225 230 CAC AAT CCA CCT CCCTTT GCT CCA CCA GTG ATG GAG TTT CCC GCT GCC 870 His Asn Pro Pro Pro PheAla Pro Pro Val Met Glu Phe Pro Ala Ala 235 240 245 CAT GAC CGC ATG GTATAC CTG GGC CTC TCA GAC TAC TTC TTC AAC ACA 918 His Asp Arg Met Val TyrLeu Gly Leu Ser Asp Tyr Phe Phe Asn Thr 250 255 260 265 GCC GGG CTT GTATAC CAA GAG GCT GGG GTC TTG AAG ATG ACC CTT AGA 966 Ala Gly Leu Val TyrGln Glu Ala Gly Val Leu Lys Met Thr Leu Arg 270 275 280 GAT GAC ATG ATTCCA AAG GAG TCC AAA TTT CGA CTG ACA ACC AAG TTC 1014 Asp Asp Met Ile ProLys Glu Ser Lys Phe Arg Leu Thr Thr Lys Phe 285 290 295 TTT GGA ACC TTCCTA CCT GAG GTG GCC AAG AAG TTT CCC AAC ATG AAG 1062 Phe Gly Thr Phe LeuPro Glu Val Ala Lys Lys Phe Pro Asn Met Lys 300 305 310 ATA CAG ATC CATGTC TCA GCC TCC ACC CCG CCA CAC CTG TCT GTG CAG 1110 Ile Gln Ile His ValSer Ala Ser Thr Pro Pro His Leu Ser Val Gln 315 320 325 CCC ACC GGC CTTACC TTC TAC CCT GCC GTG GAT GTC CAG GCC TTT GCC 1158 Pro Thr Gly Leu ThrPhe Tyr Pro Ala Val Asp Val Gln Ala Phe Ala 330 335 340 345 GTC CTC CCCAAC TCC TCC CTG GCT TCC CTC TTC CTG ATT GGC ATG CAC 1206 Val Leu Pro AsnSer Ser Leu Ala Ser Leu Phe Leu Ile Gly Met His 350 355 360 ACA ACT GGTTCC ATG GAG GTC AGC GCC GAG TCC AAC AGG CTT GTT GGA 1254 Thr Thr Gly SerMet Glu Val Ser Ala Glu Ser Asn Arg Leu Val Gly 365 370 375 GAG CTC AAGCTG GAT AGG CTG CTC CTG GAA CTG AAG CAC TCA AAT ATT 1302 Glu Leu Lys LeuAsp Arg Leu Leu Leu Glu Leu Lys His Ser Asn Ile 380 385 390 GGC CCC TTCCCG GTT GAA TTG CTG CAG GAT ATC ATG AAC TAC ATT GTA 1350 Gly Pro Phe ProVal Glu Leu Leu Gln Asp Ile Met Asn Tyr Ile Val 395 400 405 CCC ATT CTTGTG CTG CCC AGG GTT AAC GAG AAA CTA CAG AAA GGC TTC 1398 Pro Ile Leu ValLeu Pro Arg Val Asn Glu Lys Leu Gln Lys Gly Phe 410 415 420 425 CCT CTCCCG ACG CCG GCC AGA GTC CAG CTC TAC AAC GTA GTG CTT CAG 1446 Pro Leu ProThr Pro Ala Arg Val Gln Leu Tyr Asn Val Val Leu Gln 430 435 440 CCT CACCAG AAC TTC CTG CTG TTC GGT GCA GAC GTT GTC TAT AAA 1491 Pro His Gln AsnPhe Leu Leu Phe Gly Ala Asp Val Val Tyr Lys 445 450 455 TGAAGGCACCAGGGGTGCCG GGGGCTGTCA GCCGCACCTG TTCCTGATGG GCTGTGGGGC 1551 ACCGGCTGCCTTTCCCCAGG GAATCCTCTC CAGATCTTAA CCAAGAGCCC CTTGCAAACT 1611 TCTTCGACTCAGATTCAGAA ATGATCTAAA CACGAGGAAA CATTATTCAT TGGAAAAGTG 1671 CATGGTGTGTATTTTAGGGA TTATGAGCTT CTTTCAAGGG CTAAGGCTGC AGAGATATTT 1731 CCTCCAGGAATCGTGTTTCA ATTGTAACCA AGAAATTTCC ATTTGTGCTT CATGAAAAAA 1791 AACTTCTGGTTTTTTTCATG TG 1813 487 amino acids amino acid linear protein 2 Met ArgGlu Asn Met Ala Arg Gly Pro Cys Asn Ala Pro Arg Trp Val -31 -30 -25 -20Ser Leu Met Val Leu Val Ala Ile Gly Thr Ala Val Thr Ala Ala Val -15 -10-5 1 Asn Pro Gly Val Val Val Arg Ile Ser Gln Lys Gly Leu Asp Tyr Ala 510 15 Ser Gln Gln Gly Thr Ala Ala Leu Gln Lys Glu Leu Lys Arg Ile Lys 2025 30 Ile Pro Asp Tyr Ser Asp Ser Phe Lys Ile Lys His Leu Gly Lys Gly 3540 45 His Tyr Ser Phe Tyr Ser Met Asp Ile Arg Glu Phe Gln Leu Pro Ser 5055 60 65 Ser Gln Ile Ser Met Val Pro Asn Val Gly Leu Lys Phe Ser Ile Ser70 75 80 Asn Ala Asn Ile Lys Ile Ser Gly Lys Trp Lys Ala Gln Lys Arg Phe85 90 95 Leu Lys Met Ser Gly Asn Phe Asp Leu Ser Ile Glu Gly Met Ser Ile100 105 110 Ser Ala Asp Leu Lys Leu Gly Ser Asn Pro Thr Ser Gly Lys ProThr 115 120 125 Ile Thr Cys Ser Ser Cys Ser Ser His Ile Asn Ser Val HisVal His 130 135 140 145 Ile Ser Lys Ser Lys Val Gly Trp Leu Ile Gln LeuPhe His Lys Lys 150 155 160 Ile Glu Ser Ala Leu Arg Asn Lys Met Asn SerGln Val Cys Glu Lys 165 170 175 Val Thr Asn Ser Val Ser Ser Lys Leu GlnPro Tyr Phe Gln Thr Leu 180 185 190 Pro Val Met Thr Lys Ile Asp Ser ValAla Gly Ile Asn Tyr Gly Leu 195 200 205 Val Ala Pro Pro Ala Thr Thr AlaGlu Thr Leu Asp Val Gln Met Lys 210 215 220 225 Gly Glu Phe Tyr Ser GluAsn His His Asn Pro Pro Pro Phe Ala Pro 230 235 240 Pro Val Met Glu PhePro Ala Ala His Asp Arg Met Val Tyr Leu Gly 245 250 255 Leu Ser Asp TyrPhe Phe Asn Thr Ala Gly Leu Val Tyr Gln Glu Ala 260 265 270 Gly Val LeuLys Met Thr Leu Arg Asp Asp Met Ile Pro Lys Glu Ser 275 280 285 Lys PheArg Leu Thr Thr Lys Phe Phe Gly Thr Phe Leu Pro Glu Val 290 295 300 305Ala Lys Lys Phe Pro Asn Met Lys Ile Gln Ile His Val Ser Ala Ser 310 315320 Thr Pro Pro His Leu Ser Val Gln Pro Thr Gly Leu Thr Phe Tyr Pro 325330 335 Ala Val Asp Val Gln Ala Phe Ala Val Leu Pro Asn Ser Ser Leu Ala340 345 350 Ser Leu Phe Leu Ile Gly Met His Thr Thr Gly Ser Met Glu ValSer 355 360 365 Ala Glu Ser Asn Arg Leu Val Gly Glu Leu Lys Leu Asp ArgLeu Leu 370 375 380 385 Leu Glu Leu Lys His Ser Asn Ile Gly Pro Phe ProVal Glu Leu Leu 390 395 400 Gln Asp Ile Met Asn Tyr Ile Val Pro Ile LeuVal Leu Pro Arg Val 405 410 415 Asn Glu Lys Leu Gln Lys Gly Phe Pro LeuPro Thr Pro Ala Arg Val 420 425 430 Gln Leu Tyr Asn Val Val Leu Gln ProHis Gln Asn Phe Leu Leu Phe 435 440 445 Gly Ala Asp Val Val Tyr Lys 450455

I claim:
 1. A method of treating a subject suffering from infection withMycobacteria which comprises administering to the subject a compositioncomprising a BPI protein product.
 2. The method of claim 1 wherein thecomposition is administered orally.
 3. The method of claim 1 wherein thecomposition is administered intravenously.
 4. The method of claim 1wherein the composition is administered as an aerosol.
 5. The method ofclaim 1 wherein the BPI protein product is an 21-25 kD amino-terminalfragment of Bactericidal/permeability-increasing holoprotein.
 6. Themethod of claim 1 for the treatment of infection with a Mycobacteriumspecies bacterium selected from the group consisting of M. tuberculosis,M. leprae, M. intracellulare, M. avium, M. marinum, M. fortuitum, M.chelonae, M. smegmatis, M. kansasii, M. bovis, M. hominis and M.gordonae.
 7. The method of claim 1 wherein the composition furthercomprises an antibiotic.
 8. The method of claim 7 wherein the antibioticis selected from the group consisting of isoniazid, rifampin,ethambutol, p-aminosalicylic acid, pyrazinamide, streptomycin,capreomycin, cycloserine, ethionamide, kanamycin, amikacin, amithiozone,rifabutin, clofazimine, arithromycin, clarithromycin, ciprofloxacin andofloxacin.
 9. The method of claim 1 wherein the composition furthercomprises a surfactant.
 10. A method of treating a subject sufferingfrom the adverse physiological effects of the presence oflipoarabinomannan in circulation, said method comprising administeringto the subject to the subject a composition comprising a BPI proteinproduct.
 11. The method of claim 10 wherein the adverse physiologicaleffects comprise compromised immune response to microbes or tumor cellsdue to lipoarabinomannan-induced inhibition of macrophage activation byT-cell lymphokines.
 12. The method of claim 10 wherein the adversephysiological effects comprise increased production of a cytokine by thesubject.
 13. The method of claim 10 wherein the composition isadministered orally.
 14. The method of claim 10 wherein the compositionis administered intravenously.
 15. The method of claim 10 wherein thecomposition is administered as an aerosol.
 16. The method of claim 10wherein the BPI protein product is a 21-25 kD amino-terminal fragment ofBactericidal/permeability-increasing protein.
 17. The method of claim10, wherein the composition further comprises a surfactant.
 18. A methodfor decontaminating a fluid containing lipoarabinomannan said methodcomprising contacting the fluid with a BPI protein product underconditions such that lipoarabinomannan therein binds the BPI proteinproduct and separating said bound materials from said fluid.
 19. Themethod of claim 18, wherein the fluid is selected from the groupconsisting of blood, plasma, blood serum, and bone marrow.
 20. Themethod of claim 19, wherein the fluid is selected from the groupconsisting of an isotonic solution, a pharmaceutical agent, and a cellculture reagent.
 21. A pharmaceutical composition for treatment ofMycobacteria infection comprising an effective amount of a BPI proteinproduct.
 22. A pharmaceutical composition according to claim 21 furthercomprising an anti-Mycobacterial antibiotic.
 23. A pharmaceuticalcomposition for treatment of the adverse physiological effects of thepresence of lipoarabinomannan in circulation comprising a BPI proteinproduct.